Sample carrier for optical measurements

ABSTRACT

Apparatus and methods are described for determining a property of a biological sample. A sample carrier includes a glass substrate configured to define a first surface of a sample chamber that is configured to receive the sample, and a plastic substrate configured to define a second surface of the sample chamber. The height of the sample chamber at each location within the sample chamber is defined by a gap between the first surface and the second surface. An adhesive adheres the glass substrate to the plastic substrate. The plastic substrate is shaped such that the sample chamber defines a first region and a second region, with the sample chamber defining a predefined variation in height between the first region and the second region. Other applications are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/098,893 toPollak (published as US 2019/0302099), which is a US national phaseapplication of PCT Application No. PCT/IL2017/050523 to Pollak(published as WO 17/195205), filed May 11, 2017, which claims priorityfrom U.S. Provisional Patent Application No. 62/334,521 to Pollak, filedMay 11, 2016, entitled “Sample carrier for optical measurements.”

The present application is related to PCT Application No.PCT/IL2017/050526 to Zait (published as WO 17/195208), filed May 11,2017, entitled “Performing optical measurements on a sample,” whichclaims priority from U.S. Provisional Patent Application No. 62/334,517to Zait, filed May 11, 2016, entitled “Method and Apparatus forEstimating Dilution and Concentration.”

Each of the above-referenced applications is incorporated herein byreference.

FIELD OF EMBODIMENTS OF THE INVENTION

Some applications of the presently disclosed subject matter relategenerally to detecting components in a bodily sample, and in particular,to detecting components of a blood sample by performing opticalmeasurements.

BACKGROUND

In some optics-based methods (e.g., diagnostic, and/or analyticmethods), a property of a biological sample, such as a blood sample, isdetermined by performing an optical measurement. For example, thedensity of a component (e.g., a count of the component per unit volume)may be determined by counting the component within a microscopic image.Similarly, the concentration and/or density of a component may bemeasured by performing optical absorption, transmittance, fluorescence,and/or luminescence measurements upon the sample. Typically, the sampleis placed into a sample carrier and the measurements are performed withrespect to a portion of the sample that is contained within a chamber ofthe sample carrier. The measurements that are performed upon the portionof the sample that is contained within the chamber of the sample carrierare analyzed in order to determine a property of the sample.

SUMMARY OF EMBODIMENTS

In accordance with some applications of the present invention, a samplecarrier includes one or more sample chambers configured to house abiological sample (such as, a blood sample). The one or more samplechambers typically define at least first and second regions thereof, andthe height of the one or more sample chambers varies between the firstand second regions in a predefined manner. For example, the height ofthe one or more sample chambers may vary between the first and secondregions in a predefined stepped manner, or in a predefined gradualmanner.

Typically, in order to perform optical analysis upon the sample, it isdesirable to know the optical path length, the volume, and/or thethickness of the portion of the sample upon which the opticalmeasurements were performed. Further typically, optical measurements areperformed upon a portion of the sample disposed in a sample carrier thatis defined by two or more opposing surfaces (e.g., a top surface and abottom surface). In order to provide a desired level of precision fordetermining the parameter of the sample from the optical measurement, itis desirable for the two or more opposing surfaces to be separated by adistance that is correspondingly tightly set or tightly controlled.However, in some manufacture or assembly processes, the distance betweenthe opposing surfaces may vary substantially.

As described hereinabove, in accordance with some applications of thepresent invention, one or more sample chambers of a sample carrierdefine at least first and second regions thereof, and the height of theone or more sample chambers varies between the first and second regionsin a predefined manner. Typically, the sample carrier includes a firstsubstrate the defines a first surface of the one or more sample chambers(e.g. the lower surface of the one or more sample chambers), and asecond substrate that defines one or more surfaces of the one or moresample chambers that oppose the first surface (e.g. upper surfaces ofthe one or more sample chambers). The second substrate is shaped todefine the one or more surfaces that oppose the first surface, such thatone or more surfaces that oppose the first surface define the manner inwhich the height of the one or more sample chambers varies between thefirst and second regions (e.g., by defining two or more stepped surfacesthat are parallel to the first surface, and oppose the first surface).Typically, manufacturing tolerances within a single substrate, andespecially between nearby surfaces, are tighter than manufacturingtolerances on positioning between different substrates or even betweenopposing surfaces lying within the same substrate. Therefore, it istypically the case that by having a single substrate define the mannerin which the height of the one or more sample chambers varies betweenthe first and second regions, the height difference between the firstand second regions is relatively precise.

Typically, a first optical measurement is performed upon a portion ofthe sample that is disposed within the first region of the one or moresample chambers, and a second optical measurement is performed upon aportion of the sample that is disposed within the second region. Aproperty of the sample is determined by using a relationship between thefirst optical measurement, the second optical measurement, and thepredefined variation in height between the first region and the secondregion.

For some applications, a sample carrier is provided that includes one ormore sample chambers configured to house the sample. The one or moresample chambers define at least first and second regions thereof, andthe height of the one or more sample chambers varies between the firstand second regions. A biological sample is categorized and is placedinto the one or more sample chambers of the sample carrier. Based uponthe categorization of the biological sample, one of the regions of thesample carrier is selected upon which to perform optical measurementsfor measuring a given measurand. For example, if a sample, and/or amonolayer formed by the sample, has a relatively low density of redblood cells, then measurements may be performed upon a region of thesample carrier having a relatively great height, for example, such thatthere is a sufficient density of cells, and/or such that there is asufficient density of cells within the monolayer, to providestatistically reliable data. Such measurements may include, for example,red blood cell density measurements, measurements of other cellularattributes, (such as counts of abnormal red blood cells, red blood cellsthat include intracellular bodies (e.g., pathogens, Howell-Jollybodies), etc.), and/or hemoglobin concentration. Conversely, if asample, and/or a monolayer formed by the sample, has a relatively highdensity of red blood cells, then such measurements may be performed upona region of the sample carrier having a relatively low height, forexample, such that there is a sufficient sparsity of cells, and/or suchthat there is a sufficient sparsity of cells within the monolayer formedby the sample, that the cells can be identified within microscopicimages. For some applications, such methods are performed even withoutthe difference in heights between the regions being precisely known.

There is therefore provided, in accordance with some applications of thepresent invention, apparatus for determining a property of a biologicalsample, the apparatus including:

a sample carrier that includes one or more sample chambers configured tohouse the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions in a predefinedmanner; and

a computer processor configured to:

-   -   receive data relating to a first optical measurement that is        performed upon a portion of the sample that is disposed within        the first region,    -   receive data relating to a second optical measurement that is        performed upon a portion of the sample that is disposed within        the second region, and    -   determine the property of the sample by using a relationship        between the first optical measurement, the second optical        measurement, and the predefined variation in height between the        first region and the second region.

In some applications, the height of the one or more sample chambersvaries between the first and second regions in a predefined steppedmanner.

In some applications, the height of the one or more sample chambersvaries between the first and second regions in a predefined gradualmanner.

In some applications, the computer processor is configured to receivethe data relating to at least one of the first and second opticalmeasurements by receiving imaging data from a microscope.

In some applications, the computer processor is configured to receivethe data relating to at least one of the first and second opticalmeasurements by receiving data relating to a parameter selected from thegroup consisting of: optical absorption, transmittance, fluorescence,and luminescence.

In some applications, the computer processor is configured to determinethe property of the sample by determining a density of a component ofthe sample. In some applications, the computer processor is configuredto determine the property of the sample by determining a concentrationof a component of the sample. In some applications, the computerprocessor is configured to determine the property of the sample bydetermining a count of a component of the sample.

In some applications, the computer processor is configured to determinean absolute height of the one or more sample chambers within at leastone of the first and second regions, using the relationship between thefirst optical measurement, the second optical measurement, and thepredefined variation in height between the first region and the secondregion.

In some applications, the computer processor is configured to determinethe property of the sample, by:

subtracting a parameter derived from the first optical measurement froma parameter derived from the second optical measurement; and

determining the property of the sample, based upon a relationshipbetween a result of the subtracting and the predefined variation inheight between the first region and the second region.

In some applications, the computer processor is configured to determinethe property of the sample, by:

dividing a parameter derived from the second optical measurement by aparameter derived from the first optical measurement; and

determining the property of the sample, based upon a relationshipbetween a result of the dividing and the predefined variation in heightbetween the first region and the second region

In some applications, the biological sample includes a blood sample, andthe computer processor is configured to determine the property of thebiological sample by determining a property of the blood sample. In someapplications, the computer processor is configured to determine theproperty of the sample by determining a concentration of a givencomponent within the blood sample. In some applications, the computerprocessor is configured to determine the property of the sample bydetermining a count of a given component within the blood sample. Insome applications, the computer processor is configured to determine theproperty of the sample by determining a density of a given componentwithin the blood sample.

In some applications, the one or more sample chambers define at leastfirst, second, and third regions thereof, the height of the one or moresample chambers varying between each of the first, second, and thirdregions in a predefined manner.

In some applications, the computer processor is configured to:

receive data relating to a third optical measurement that is performedupon a portion of the sample that is disposed within the third region;and

to determine the property of the sample, by performing statisticalanalysis with respect to the first, second, and third opticalmeasurements, and the predefined variation in height between the first,second, and third regions.

In some applications, the computer processor is configured to:

determine a signal level of the biological sample, and

based upon the determined signal level, select two out of the first,second, and third regions upon which to perform, respectively, the firstand second optical measurements.

In some applications, the sample carrier includes:

a first substrate that defines a first surface; and

a second substrate that defines one or more surfaces that oppose thefirst surface, and

the second substrate is shaped to define the one or more surfaces thatoppose the first surface, such that one or more surfaces that oppose thefirst surface define the manner in which the height of the one or moresample chambers varies between the first and second regions.

In some applications, the second substrate defines second and thirdsurfaces, the second and third surfaces (a) opposing the first surface,(b) being parallel to the first surface, and (c) being stepped withrespect to each other. In some applications, the second substrate thatdefines at least a second surface, the second surface (a) opposing thefirst surface, and (b) being non-parallel with respect to the firstsurface.

There is further provided, in accordance with some applications of thepresent invention, a method for determining a property of a biologicalsample, the method including:

providing a sample carrier, the sample carrier including one or moresample chambers configured to house the sample, the one or more samplechambers defining at least first and second regions thereof, a height ofthe one or more sample chambers varying between the first and secondregions in a predefined manner;

placing the sample into the one or more sample chambers;

performing a first optical measurement upon a portion of the sample thatis disposed within the first region;

performing a second optical measurement upon a portion of the samplethat is disposed within the second region; and

determining the property of the sample by using a relationship betweenthe first optical measurement, the second optical measurement, and thepredefined variation in height between the first region and the secondregion.

There is further provided, in accordance with some applications of thepresent invention, a computer software product, for use with abiological sample that is placed within a sample carrier that includesone or more sample chambers configured to house the sample, the one ormore sample chambers defining at least first and second regions thereof,a height of the one or more sample chambers varying between the firstand second regions, in a predefined manner, the computer softwareproduct including a non-transitory computer-readable medium in whichprogram instructions are stored, which instructions, when read by acomputer cause the computer to perform the steps of:

receiving data relating to a first optical measurement that is performedupon a portion of the sample that is disposed within the first region;

receiving data relating to a second optical measurement that isperformed upon a portion of the sample that is disposed within thesecond region; and

determining the property of the sample by using a relationship betweenthe first optical measurement, the second optical measurement, and thepredefined variation in height between the first region and the secondregion.

There is further provided, in accordance with some applications of thepresent invention, a method performing optical measurements on abiological sample, the method including:

providing a sample carrier that includes one or more sample chambersconfigured to house the sample, the one or more sample chambers definingat least first and second regions thereof, a height of the one or moresample chambers varying between the first and second regions;

categorizing the biological sample;

placing the sample into the one or more sample chambers; and

based upon the categorization of the biological sample, selecting one ofthe first and second regions upon which to perform optical measurementsfor measuring a given measurand.

In some applications, categorizing the sample includes receiving anindication of the categorization of the sample. In some applications,categorizing the sample includes categorizing the sample based upon adensity of one or more components within the sample. In someapplications, categorizing the sample includes categorizing the samplebased upon a surface density of one or more components within amonolayer formed by the sample. In some applications, categorizing thesample includes categorizing the sample based upon a concentration ofone or more components within the sample. In some applications,categorizing the sample includes categorizing the sample based upon acount of one or more components within the sample. In some applications,categorizing the sample includes measuring a parameter of the sampleselected from the group consisting of: optical absorption,transmittance, fluorescence, and luminescence, by performing apreliminary optical measurement upon the sample. In some applications,categorizing the sample includes performing microscopic imaging upon thesample.

In some applications, selecting one of the first and second regions uponwhich to perform optical measurements for measuring the given measurandincludes selecting one of the first and second regions upon which toperform counting of a given component within the sample, by performingmicroscopic imaging upon the region. In some applications, selecting oneof the first and second regions upon which to perform opticalmeasurements for measuring the given measurand includes selecting one ofthe first and second regions upon which to measure a concentration of agiven component within the sample, by measuring a parameter selectedfrom the group consisting of: optical absorption, transmittance,fluorescence, and luminescence.

In some applications:

the one or more sample chambers define at least first, second, and thirdregions thereof, a height of the one or more sample chambers varyingbetween each of the first, second, and third regions in a predefinedmanner; and

based upon the identified property of the biological sample, selectingtwo out of the first, second, and third regions upon which to perform,respective, first and second optical measurements for measuring thegiven measurand.

In some applications, the method further includes:

performing the, respective, first and second optical measurements uponthe selected two regions; and

measuring the given measurand by using a relationship between the firstoptical measurement, the second optical measurement, and the predefinedvariation in height between the selected two regions.

In some applications, the biological sample includes a blood sample, andselecting one of the first and second regions upon which to performoptical measurements for measuring the given measurand includesselecting one of the first and second regions upon which to performoptical measurements for measuring a given measurand of the bloodsample.

In some applications, selecting one of the first and second regions uponwhich to perform optical measurements for measuring the given measurandincludes selecting one of the first and second regions upon which tomeasure a concentration of a given component within the blood sample, bymeasuring a parameter selected from the group consisting of: opticalabsorption, optical transmittance, fluorescence, and luminescence. Insome applications, selecting one of the first and second regions uponwhich to perform optical measurements for measuring the given measurandincludes selecting one of the first and second regions upon which toperform counting of a given component within the blood sample, byperforming microscopic imaging upon the region.

There is further provided, in accordance with some applications of thepresent invention, apparatus for determining a property of a biologicalsample, the apparatus including:

a sample carrier that includes one or more sample chambers configured tohouse the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions; and

a computer processor configured to:

-   -   categorize the biological sample, and    -   based upon the categorization of the biological sample, select        one of the first and second regions upon which to perform        optical measurements for measuring a given measurand of the        biological sample.

There is further provided, in accordance with some applications of thepresent invention, a computer software product, for use with abiological sample that is placed within a sample carrier that includesone or more sample chambers configured to house the sample, the one ormore sample chambers defining at least first and second regions thereof,a height of the one or more sample chambers varying between the firstand second regions, the computer software product including anon-transitory computer-readable medium in which program instructionsare stored, which instructions, when read by a computer cause thecomputer to perform the steps of:

categorizing the biological sample; and

based upon the categorization of the biological sample, selecting one ofthe first and second regions upon which to perform optical measurementsfor measuring a given measurand of the biological sample.

There is further provided, in accordance with some applications of thepresent invention, apparatus for performing optical measurements on abiological sample, the apparatus including:

a sample carrier that includes one or more sample chambers configured tohouse the sample,

the one or more sample chambers defining at least first, second, andthird regions thereof, a height of the one or more sample chambersvarying between each of the first, second, and third regions in apredefined manner.

There is further provided, in accordance with some applications of thepresent invention, a method for performing optical measurements on abiological sample, the method including:

providing a sample carrier, the sample carrier including one or moresample chambers configured to house the sample, the one or more samplechambers defining at least first and second regions thereof, a height ofthe one or more sample chambers varying between the first and secondregions;

placing the sample into the one or more sample chambers;

measuring a first measurand, by performing a first optical measurementupon a portion of the sample that is disposed within the first region;and

measuring a second measurand, by performing a second optical measurementupon a portion of the sample that is disposed within the second region.

In some applications, the biological sample includes a blood sample,measuring the first measurand includes measuring a first measurand ofthe blood sample performing the first optical measurement upon a portionof the blood sample that is disposed within the first region, andmeasuring the second measurand includes measuring a second measurand ofthe blood sample by performing a second optical measurement upon aportion of the sample that is disposed within the second region.

In some applications, measuring the first measurand of the blood sampleincludes determining a count of a first component within the bloodsample by performing microscopic imaging upon the portion of the samplethat is disposed within the first region, and measuring the secondmeasurand of the blood sample includes determining a count of a secondcomponent within the blood sample by performing microscopic imaging uponthe portion of the sample that is disposed within the second region.

In some applications, measuring the first measurand of the blood sampleincludes measuring a concentration of a first component within the bloodsample, by performing, upon the portion of the sample that is disposedwithin the first region, an optical measurement of a parameter selectedfrom the group consisting of: optical absorption, transmittance,fluorescence, and luminescence. In some applications, measuring thesecond measurand of the blood sample includes measuring a concentrationof a second component within the blood sample, by performing, upon theportion of the sample that is disposed within the second region, anoptical measurement of a parameter selected from the group consistingof: optical absorption, transmittance, fluorescence, and luminescence.In some applications, measuring the second measurand of the blood sampleincludes determining a count of a second component within the bloodsample by performing microscopic imaging upon the portion of the samplethat is disposed within the second region.

There is further provided, in accordance with some applications of thepresent invention, apparatus for determining a property of a biologicalsample, the apparatus including:

a sample carrier that includes one or more sample chambers configured tohouse the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions; and

a computer processor configured to:

-   -   measure a first measurand, by receiving a first optical        measurement performed upon a portion of the sample that is        disposed within the first region; and    -   measure a second measurand, by receiving a second optical        measurement performed upon a portion of the sample that is        disposed within the second region.

There is further provided, in accordance with some applications of thepresent invention, a computer software product, for use with abiological sample that is placed within a sample carrier that includesone or more sample chambers configured to house the sample, the one ormore sample chambers defining at least first and second regions thereof,a height of the one or more sample chambers varying between the firstand second regions, the computer software product including anon-transitory computer-readable medium in which program instructionsare stored, which instructions, when read by a computer cause thecomputer to perform the steps of:

measuring a first measurand, by receiving a first optical measurementperformed upon a portion of the sample that is disposed within the firstregion; and

measuring a second measurand, by receiving a second optical measurementperformed upon a portion of the sample that is disposed within thesecond region.

There is further provided, in accordance with some applications of thepresent invention, a method performing optical measurements on abiological sample, the method including:

providing a sample carrier that includes one or more sample chambersconfigured to house the sample, the one or more sample chambers definingat least first and second regions thereof, a height of the one or moresample chambers varying between the first and second regions;

categorizing a measurand of the biological sample that is to bemeasured;

placing the sample into the one or more sample chambers; and

based upon the categorization of the measurand, selecting one of thefirst and second regions upon which to perform optical measurements formeasuring the identified measurand.

In some applications, the biological sample includes a blood sample,categorizing the measurand of the biological sample that is to bemeasured includes categorizing a measurand of the blood sample that isto be measured.

In some applications, selecting one of the first and second regions uponwhich to perform optical measurements for measuring the identifiedmeasurand includes selecting one of the first and second regions uponwhich to measure a concentration of a given component within the bloodsample, by measuring a parameter selected from the group consisting of:optical absorption, transmittance, fluorescence, and luminescence. Insome applications, selecting one of the first and second regions uponwhich to perform optical measurements for measuring the identifiedmeasurand includes selecting one of the first and second regions uponwhich to perform microscopic imaging.

There is further provided, in accordance with some applications of thepresent invention, apparatus for determining a property of a biologicalsample, the apparatus including:

a sample carrier that includes one or more sample chambers configured tohouse the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions; and

a computer processor configured to:

-   -   categorize a measurand of the biological sample that is to be        measured, and    -   based upon the categorization of the measurand, select one of        the first and second regions upon which to perform optical        measurements for measuring the identified measurand.

There is further provided, in accordance with some applications of thepresent invention, a computer software product, for use with abiological sample that is placed within a sample carrier that includesone or more sample chambers configured to house the sample, the one ormore sample chambers defining at least first and second regions thereof,a height of the one or more sample chambers varying between the firstand second regions, the computer software product including anon-transitory computer-readable medium in which program instructionsare stored, which instructions, when read by a computer cause thecomputer to perform the steps of:

categorizing a measurand of the biological sample that is to bemeasured; and

based upon the categorization of the measurand, selecting one of thefirst and second regions upon which to perform optical measurements formeasuring the identified measurand.

The present invention will be more fully understood from the followingdetailed description of embodiments thereof, taken together with thedrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing components of a biological sampleanalysis system, in accordance some applications of the presentinvention;

FIG. 2 is a schematic cross-sectional illustration of a sample carrierthat defines a variation in height that is stepped, in accordance withsome applications of the present invention;

FIG. 3 is a schematic cross-sectional illustration of a sample carrierthat defines a variation in height that is gradual, in accordance withsome applications of the present invention;

FIG. 4 is a schematic cross-sectional illustration of a sample carrierthat includes one or more sample chambers that define first, second, andthird regions, the height of the one or more sample chambers varyingbetween each of the first, second, and third regions in a predefinedmanner, in accordance with some applications of the present invention;

FIG. 5 is a flowchart showing steps of algorithm that is performed inaccordance with some applications of the present invention; and

FIG. 6 is a flowchart showing steps of algorithm that is performed inaccordance with some applications of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Reference is now made to FIG. 1, which is block diagram showingcomponents of a biological sample analysis system 20, in accordance someapplications of the present invention. Typically, a biological sample(e.g., a blood sample) is placed into a sample carrier 22. While thesample is disposed in the sample carrier, optical measurements areperformed upon the sample using one or more optical measurement devices24. For example, the optical measurement devices may include amicroscope (e.g., a digital microscope), a spectrophotometer, aphotometer, a spectrometer, a camera, a spectral camera, a hyperspectralcamera, a fluorometer, a spectrofluorometer, and/or a photodetector(such as a photodiode, a photoresistor, and/or a phototransistor). Forsome applications, the optical measurement devices include dedicatedlight sources (such as light emitting diodes, incandescent lightsources, etc.) and/or optical elements for manipulating light collectionand/or light emission (such as lenses, diffusers, filters, etc.). Forsome applications, a microscope system is used that is generally similarto the microscope system described in US 2014/0347459 to Greenfield,which is incorporated herein by reference.

A computer processor 28 typically receives and processes opticalmeasurements that are performed by the optical measurement device.Further typically, the computer processor controls the acquisition ofoptical measurements that are performed by the one or more opticalmeasurement devices. The computer processor communicates with a memory30. A user (e.g., a laboratory technician) sends instructions to thecomputer processor via a user interface 32. For some applications, theuser interface includes a keyboard, a mouse, a joystick, a touchscreendevice (such as a smartphone or a tablet computer), a touchpad, atrackball, a voice-command interface, and/or other types of userinterfaces that are known in the art. Typically, the computer processorgenerates an output via an output device 34. Further typically, theoutput device includes a display, such as a monitor, and the outputincludes an output that is displayed on the display. For someapplications, the processor generates an output on a different type ofvisual, text, graphics, tactile, audio, and/or video output device,e.g., speakers, headphones, a smartphone, or a tablet computer. For someapplications, user interface 32 acts as both an input interface and anoutput interface, i.e., it acts as an input/output interface. For someapplications, the processor generates an output on a computer-readablemedium (e.g., a non-transitory computer-readable medium), such as adisk, or a portable USB drive, and/or generates an output on a printer.

Reference is now made to FIG. 2, which is a schematic cross-sectionalillustration of sample carrier 22, in accordance with some applicationsof the present invention. The sample carrier defines one or more samplechambers 40, into which the sample is placed. The one or more samplechambers typically define at least a first region 42 (which isshallower) and a second region 44 (which is deeper), the height of theone or more sample chambers varying between the first and second regionsin a predefined manner. For example, as shown in FIG. 2, the height ofthe one or more sample chambers varies between the first and secondregions in a predefined stepped manner.

Typically, in order to perform optical analysis upon the sample, it isdesirable to know the optical path length, the volume, and/or thethickness of the portion of the sample upon which the opticalmeasurements were performed, as precisely as possible. Furthertypically, the optical measurements are performed upon a portion of thesample disposed in a sample carrier that is defined by two or moreopposing surfaces. In order to provide the desired level of precision,it is desirable for the two or more opposing surfaces to be separated bya distance that is correspondingly tightly set or tightly controlled.However, in some manufacture or assembly processes, the distance betweenthe opposing surfaces may vary substantially. For example, in someinstances, two or more of the opposing surfaces lie in separatesubstrates that are bonded relative to each other during manufacture orassembly (e.g. using thermal bonding, solvent-assisted bonding,ultrasonic welding, laser welding, heat staking, adhesive, mechanicalclamping and/or additional substrates).

For example, as shown in FIG. 2, the height of first region 42 of thesample chamber is defined by a lower surface 46 that is defined by afirst substrate 48 (e.g., a glass or a plastic substrate) and by uppersurface 50 that is defined by a second substrate 52 (e.g., a plasticsubstrate, such as an injection-molded plastic substrate). The first andsecond substrates are bonded to each other by an adhesive layer 53,e.g., a pressure-sensitive adhesive. Examples of the adhesive layerinclude an additional physical layer (such as a pressure-sensitiveadhesive layer), a sandwich of pressure-sensitive adhesive and a carrierlayer (such as a polyethylene terephthalate layer), a bonding layer(such as a solvent-assisted bonding layer), or a layer resulting from aprocess performed upon the top and bottom substrates (such as ultrasonicwelding) without necessarily introducing additional materials or pieces.Although the adhesive layer has a nominal thickness, it is typically thecase that, for example, due to variation in the manufactured thicknessof the pressure-sensitive adhesive or in the pressure applied during itsapplication, the actual thickness of the layer is different from thenominal thickness. For example, the two substrates may be bonded using apressure-sensitive adhesive layer with a nominal thickness that isconfigured to separate the opposing surfaces by a separation of 100micrometers. In such a case, variation in the manufactured thickness ofthe pressure-sensitive adhesive layer or in the pressure applied duringits application may result in a final thickness that may lie, forexample, as far as 20 micrometers greater or less than the nominalthickness.

Typically, an optical measurement is performed on the sample. Forexample, the density of a component may be determined by performing acount of the component within a microscopic image. Similarly, theconcentration and/or density of a component may be measured byperforming optical absorption, transmittance, fluorescence, and/orluminescence measurements upon the sample. Without being bound bytheory, an uncertainty of 20 percent in the distance separating the twoopposing surfaces (as described in the above example), may, in turn,correspond to 20 percent uncertainty in parameters of the sample thatare derived from the optical measurements that are performed upon thesample (such as, the derived concentration and/or density of a componentwithin the sample).

For example, for some applications, the concentration of a component isdetermined by measuring optical absorption. The absorption measurementsare analyzed based upon the Beer-Lambert Law, in accordance with whichthe resulting optical intensity I after passing through a distance h ina sample containing concentration ρ of a substance with absorptivitycoefficient α is I=I₀×e^(−αρh), where I₀ is incident the light intensitybefore passing through the sample. Thus, for some applications, whenpassing light through a sample within a sample chamber having a height h(which is defined by the distance between the opposing surfaces), I andI₀ are measured and the concentration of a given component is deducedusing the known height and the known absorptivity coefficient of thecomponent. For example, such a technique may be used to measure thehemoglobin concentration of a blood sample (e.g., using absorptiontechniques that are known in the art, such as, by first staininghemoglobin using a suitable dye that provides an optical absorptionsignature, or by performing the measurements upon unstained hemoglobin).For some applications, additional measurements are performed atdifferent wavelengths to further improve the accuracy in determining theconcentration. For such techniques, uncertainty in the height h of thesample chamber results in a corresponding uncertainty in the derivedconcentration.

For some applications, the density (e.g. count per unit volume) of acomponent is measured. For example, such measurements may be performedin order to count the number of red blood cells, white blood cells,platelets, reticulocytes, Howell-Jolly bodies, bacteria, and/orparasites of a given type per unit volume, such as when performing acomplete blood count or a diagnostic test. Typically, for suchapplications, images (e.g., microscopic images) of the sample areacquired, and the count per unit volume is determined based upon thecount of the component within the images and the corresponding volumewithin which the count was measured. As the volume is equal to heighttimes area, any uncertainty in the height of the sample chamber resultsin uncertainty in the volume, and a corresponding uncertainty in thecount per unit volume.

For some applications, one or more of the following measurements areperformed upon a sample within a sample chamber: bacteria or virusconcentration, contaminant concentration (e.g. in drinking water),turbidity measurement (e.g. in water, urine), and enzymatic assays(including enzyme-linked immunosorbent assays). For such measurements,uncertainty in the height of the sample chamber results in uncertaintyin the measurement

In accordance with some applications of the present invention, theabove-described problems associated with uncertainty relating to theheight of a sample chamber are at least partially overcome. Referringagain to FIG. 2, sample chamber defines first region 42 and a secondregion 44. The height of first region 42 of the sample chamber isdefined by lower surface 46 that is defined by first substrate 48 and byupper surface 50 that is defined by second substrate 52. The height ofsecond region 44 of the sample chamber is defined by lower surface 46and by second upper surface 54 that is defined by second substrate 52.As shown second upper surface 54 is stepped with respect to first uppersurface 50, and both surfaces 50 and 54 are parallel to lower surface46. The first and second substrates are bonded to each other by adhesivelayer 53, e.g., a pressure-sensitive adhesive, such that absolute heighth of the first region 42 (which is shallower) is uncertain, e.g., forthe reasons described hereinabove. The step between first upper surface50 and second upper surface 54, provides a predefined height differenceΔh between the first, shallower region and the second, deeper region,such that even though height h of the first region is not known to asufficient degree of accuracy, the height difference Δh is known to asufficient degree of accuracy to determine a parameter of the sample,using the techniques described herein.

As shown in FIG. 2, second substrate 52 is shaped to define surfaces 50and 54, such that surfaces 50 and 54 define the manner in which theheight of the one or more sample chambers varies between the first andsecond regions. Typically, relative manufacturing tolerances within asingle substrate, and especially between nearby surfaces, are tighterthan manufacturing tolerances on positioning between differentsubstrates or even between opposing surfaces lying within the samesubstrate. Therefore, it is typically the case that by having a singlesubstrate define the manner in which the height of the one or moresample chambers varies between the first and second regions, the heightdifference between the first and second regions is relatively precise.For example, second substrate 52 may be manufactured with relativelytight tolerances, for example, using injection molding, embossing ormachining.

An illustrative example of how the height difference Δh may be used todetermine a parameter of the sample is as follows. In order to determinethe density of white blood cells within a blood sample, the number ofwhite blood cells within a microscopic image within a given area Awithin region 42 may be counted, and the number of white blood cellswithin the same area within region 44 may also be counted. Thedifference between these two numbers is equal to the number of whiteblood cells in a volume equal to area A multiplied by height differenceΔh. Therefore, the number of white blood cells within this volume isdivided by the known volume, to provide the density of white blood cellsper unit volume in the solution that is disposed in the carrier.Typically, this value is used to extrapolate an amount or concentrationof white blood cells in a stock sample, from which the solution in thesample carrier was produced.

For some applications, additional steps are performed to reduce theerror in estimating the white blood cell density. For example, a choiceof height differences may be provided, such that a suitable heightdifference is chosen, and/or such that measurements obtained acrossmultiple height differences are integrated using a statistical method(e.g. averaging, regression, curve-fitting or other techniques known inthe art). For some applications, the above-described technique isperformed but with different areas being measured in regions 42 and 44,and with the volume being calculated by correcting for the areadifference between the areas that were measured in regions 42 and 44.

For some applications, the above-described technique is used todetermine the density (e.g., the count per unit volume) of othercomponents within a blood sample, including but limited to red bloodcells, platelets, anomalous white blood cells, circulating tumor cells,reticulocytes, Howell Jolly bodies, pathogens (such as, Plasmodium orBabesia), etc.

It is noted that although height h of first, shallower region 42 isshown in FIG. 2 as being defined solely be the thickness of adhesivelayer 53, the height h may be defined by a combination of the adhesionlayer and protrusions or extrusions from first substrate 48 and/orsecond substrate 52. For some applications, the thickness of theadhesive layer varies not only between sample carriers, but even in thesame carrier or even along a single sample chamber. Such variation mayaffect absolute height h of the first region or the height differencebetween the first and second regions. If this variation is known inadvance, it is typically factored into calculations that are performedupon the optical measurements. Typically, the variation in the thicknessof the adhesive layer is less than 10 percent along the regions uponwhich the optical measurements are performed.

It is further noted that, although in FIG. 2 regions 42 and 44 are shownas being regions within a single sample chamber without any separationbetween the two regions, for some applications, regions 42 and 44 areregions within respective sample chambers that are at least partiallyseparated from each other. In accordance with some applications, thechambers are positioned adjacent to one another, in a linear array, orin any regular lattice shape. For some applications, the chambers areseparated from one another by an adhesive layer or a spacer. For someapplications, the chambers are positioned adjacent to one another andare filled with the sample using capillary forces. Typically, for suchapplications, the sample is inserted into the sample carrier via anentry hole, and the sample chamber defines an air exit hole via whichair exits the sample carrier, in order to facilitate filling of thesample carrier with the sample. For some such applications, the samplechambers are arranged such that the chamber that has the greatest heightis closest to the entry hole, and the sample chamber that has the lowestheight is closest to the to the air exit hole, with any additionalchambers being arranged in corresponding height order.

For some applications, an optical measurement is performed by providingoptical windows on the sample carrier. For example, absorptionmeasurements may be performed by illuminating a sample through a regionof one of the substrates (e.g., top substrate 52) that defines anoptical window 60 and measuring light coming out through a region of theother substrate (e.g. bottom substrate 48) that defines an opticalwindow 62. For some applications, a reflective surface is used to allowthe light to enter and exit through the same optical window (e.g.,window 60). This may be used, for example, in the case of an absorptionor density measurement, with the analysis having to account, forexample, for light having gone through the sample twice. For someapplications fluorescence is measured using one or more optical windows.For example, epifluorescence measurements may be performed through asingle optical window, since the emitted light may be detected throughthe same optical window as used for excitation light. For someapplications, luminescence is measured using one or more opticalwindows.

Although FIG. 2 shows only upper substrate 52 defining the steppedsurfaces, for some applications, both the upper and lower substratesdefine surfaces that are stepped with respect to one another (or theyboth define a surface that is sloped or curved, as describedhereinbelow). For some applications, only the lower substrate definessurfaces that are stepped with respect to one another (or defines asurface that is sloped or curved, as described hereinbelow). For someapplications, measurements are performed in a lateral direction, withrespect to the sample carrier, and a substrate that defines the lateralsurfaces of the one or more sample chambers defines surfaces that arestepped with respect to one another (or defines a surface that is slopedor curved, as described hereinbelow).

Reference is now made to FIG. 3, which is a schematic illustration ofsample carrier 22, in accordance with some applications of the presentinvention. Sample carrier 22 as shown in FIG. 3 is generally similar tosample carrier 22 as described hereinabove, expect that second substrate52 is shaped such that the height of the one or more sample chambersvaries in a gradual manner. As shown, for some applications, a singlesloped surface 64 defined by second substrate 52 defines the manner inwhich the height of the one or more sample chambers varies. For suchapplications, the height difference between first and second regionsupon which optical measurements are performed is determined based uponthe predefined slope of the surface and the relative spacing of thefirst and second regions upon which the measurements are performed. Forsome applications, differently shaped surfaces defined by secondsubstrate 52 define the manner in which the height of the one or moresample chambers varies between the first and second regions. Forexample, a curved surface may be used, which may allow measurements witha larger height difference to be taken in one region versus anotherregion.

Reference is now made to FIG. 4, which is a schematic cross-sectionalillustration of sample carrier 22, the sample carrier including one ormore sample chambers that define first region 42, second region 44, anda third region 66, the height of the one or more sample chambers varyingbetween each of the first, second, and third regions in a predefinedmanner, in accordance with some applications of the present invention.Sample carrier is generally as described hereinabove, except for thedifferences described below. As shown, the height of the second regionis greater than the height of the first region by a height differenceΔh1, and the height of the third region is greater than the height ofthe second region by a height difference Δh2 (such that the height ofthe third region is greater than the height of the first region by aheight difference (Δh1+Δh2)) The height differences between the regionsare defined by three surfaces 50, 54, and 66 defined by second substrate52, each of the three surfaces opposing surface 46, defined by firstsubstrate 48. It is noted that the scope of the present inventionincludes using a sample carrier that defines more than three regions(e.g., 4-10 regions) having predefined height differences between them,and which are defined by a single substrate, mutatis mutandis.

Reference is now made to FIG. 5, which is a flowchart showing steps ofalgorithm that is performed by computer processor 28, in accordance withsome applications of the present invention. The flowchart is describedwith reference to the sample carrier shown in FIG. 2, but the algorithmcould be applied to other embodiments of the sample carrier as describedherein, mutatis mutandis. For some applications, in order to determinethe concentration of a given component within a biological sample thatis disposed within sample carrier 22, light is transmitted throughregions 42 and 44. The light intensity detected after transmissionthrough these regions is detected by optical measurement device 24, andthese light intensity measurements are received by computer processor instep 70. In step 72, for each of these measurements, the detected lightintensity is divided by the incident light intensity. In step 74, thenatural logarithm of the outputs of step 72 is calculated. In step 76,the outputs of step 74 are divided by the absorptivity coefficient ofthe component being measured, which provides ρ×h for region 42 andρ×(h+Δ_(h)) for region 44. In step 78, the output of step 76 for region42 is subtracted from the output of step 76 for region 44, whichprovides ρΔ_(h). In step 80, the output of step 78 is divided by theknown height difference, to provide the concentration ρ.

For some applications, three or more regions having a known heightvariation between them are used (e.g., using a sample chamber as shownin FIG. 4), and the algorithm shown in FIG. 5 is repeated with respectto respective pairs of regions with known height differences betweenthem, such that the concentration of the component is determined usingdifferent combinations of measured light intensities. Typically,measurements obtained across multiple height differences are integratedusing a statistical method (e.g. averaging, regression, curve-fitting orother techniques known in the art), in order to provide a finaldetermination of the concentration of the component. For someapplications, discrepancies between the different measurements are usedas an indication that there are errors in the measurement or that thesample preparation was not performed correctly (e.g., due tounsuccessful filling of the sample carrier, resulting in remainingbubbles, or untreated blood, etc.). For some applications, in responsethereto, a sample is rejected from being used, and/or the computerprocessor determines that the results obtained for the sample should betreated with a decreased level of confidence relative to other samplesor portions thereof, and a corresponding indication is generated uponthe output device.

For some applications, the intensity of light that is reflected from thesample is measured, rather than measuring light that is transmitted fromthe sample. For such applications, the algorithm described withreference to FIG. 5 is modified accordingly.

For some applications, similar techniques are applied to opticalmeasurements that relate to fluorescence or luminescence opticalsignatures. For example, the detected luminescence of a sample may beproportional to the volume assayed by an optical detector, which in turnmay be proportional to sample height. The techniques described hereinallow a practitioner to perform the measurement in two or more separateregions of the device that have predefined height differencestherebetween. For some applications, the height differences are known toa greater degree of accuracy than the overall height of the samplechamber, as described hereinabove. For some applications, the heightdifferences are used, for example, to mathematically infer sampleluminescence per unit volume, which in turn may be used to assess theconcentration, count or density of a component of the sample.

For some application, the techniques described with reference to FIG. 5are used in order to determine the concentration of hemoglobin and/orother components within a blood sample.

As described hereinabove, for some applications concentration isdetermined by comparing the light intensity before passing through thesample to the measured light intensity after light has been transmittedthrough, or reflected by, the sample. As the measured light intensitymay be up to a few orders of magnitude smaller than the transmittedlight intensity, this may require the ability to provide accurate lightintensity measurements over a large dynamic range of measuredintensities. Alternatively, one may provide the incident light andmeasure the transmitted or reflected light at a range of differentemitter or detector settings, in which case this may require preciseknowledge of how the emitter or detector behavior changes with changingthe settings (e.g. how emitted light intensity varies with inputcurrent).

For some applications of the present invention, the concentration of agiven component within the sample is determined without requiringknowledge of the intensity of the transmitted light intensity, bycomparing measured light intensities corresponding to respective regionswithin the sample carrier, and without changing the intensity of theincident light between measurements. For example, with reference to thesample carrier as shown in FIG. 2, if the measured intensity of lighttransmitted through region 42 is defined as I_(h) and the measuredintensity of light transmitted through region 42 is defined as I_(h+Δ) ₁, the concentration of a given component ρ is given by:

$\rho = {\frac{1}{\alpha \Delta_{1}}\log {\frac{I_{h}}{I_{h + \Delta_{1}}}.}}$

For some such applications, the actual system setting used is chosensuch as to provide desirable operating conditions.

For some applications, sample carrier 22 defines three or more regionswith predefined height differences between them, for example, as shownin FIG. 4. For some applications, the regions upon which to perform themeasurements are selected, based upon the concentration of one or morecomponents within the sample that is being analyzed, such as to providea dynamic range of concentrations of the sample that can be measured.For example, for lower concentrations of the sample, absorption througha larger optical length may be measured, while for higher concentrationsof the sample, absorption through a smaller optical length may bemeasured. In order to provide a range of optical lengths via whichmeasurements can be performed, the sample carrier may be shaped todefine several regions having different height differences between them(e.g., a second region being greater in height than a first region by 30micrometers, a third region being greater in height than the secondregion by 60 micrometers, a fourth region being greater in height thanthe third region by 120 micrometers, etc.). Alternatively, the samplecarrier may be shaped to define several repetitions of the same or asimilar height difference (e.g., a second region being greater in heightthan a first region by 30 micrometers, a third region being greater inheight than the second region by 30 micrometers, a fourth region beinggreater in height than the third region by 30 micrometers, etc.). In thelatter case, for low concentration of the sample, one would choose whichcombination of regions to use, such as to provide a suitable heightdifference, based upon the concentration of one or more componentswithin the sample. For some applications, the regions upon whichmeasurements are performed are chosen to provide repeated measurementsat the same height difference, or to provide a plurality of measurementsat different height differences. For some applications, measurementsobtained across multiple height differences are integrated using astatistical method (e.g. averaging, regression, curve-fitting or othertechniques known in the art), in order to provide a final determinationof the concentration of a component.

In general, the scope of the present invention includes (a) providing asample carrier, such as sample carrier 22 as described herein, (b)categorizing a biological sample, (c) placing the sample into the one ormore sample chambers of the sample carrier, and (d) based upon thecategorization of the biological sample, selecting one of the regions ofthe sample carrier upon which to perform optical measurements formeasuring a given measurand. For example, if a sample, and/or amonolayer formed by the sample, has a relatively low density of redblood cells, then measurements may be performed upon a region of thesample carrier having a relatively great height, such that there is asufficient density of cells, and/or such that there is a sufficientdensity of cells within the monolayer formed by the sample, to providestatistically reliable data. Such measurements may include, for examplered blood cell density measurements, measurements of other cellularattributes, (such as counts of abnormal red blood cells, red blood cellsthat include intracellular bodies (e.g., pathogens, Howell-Jollybodies), etc.), and/or hemoglobin concentration. Conversely, if asample, and/or a monolayer formed by the sample, has a relatively highdensity of red blood cells, then such measurements may be performed upona region of the sample carrier having a relatively low height, forexample, such that there is a sufficient sparsity of cells, and/or suchthat there is a sufficient sparsity of cells within the monolayer ofcells formed by the sample, that the cells can be identified withinmicroscopic images. For some applications, such methods are performedeven without the variation in height between the regions of the one ormore sample chambers being precisely known.

For some applications, the sample is categorized based on receiving anindication of the categorization of the sample (e.g., the sample may belabelled to indicate its categorization and this categorization may beinputted into the computer processor). Alternatively or additionally,the categorization includes performing microscopic imaging upon thesample, and/or measuring a parameter of the sample, such as opticalabsorption, transmittance, fluorescence, and/or luminescencemeasurements, by performing a preliminary optical measurement upon thesample. For some applications, the sample is categorized based on theconcentration of one or more components within the sample, and/or basedon the density (e.g., a count per unit volume) of one or more componentswithin the sample. For some applications, a monolayer is formed withinthe sample carrier (for example, using techniques as described in U.S.Pat. No. 9,329,129 to Pollak, which is incorporated herein byreference), and the sample is categorized based upon a surface densityof one or more components of the sample within the monolayer.

For some applications, based upon the measurand that is being measured,the region within the sample carrier upon which to perform opticalmeasurements is selected. For example, a region of the sample chamberhaving a relatively great height may be used to perform a white bloodcell count (e.g., to reduce statistical errors which may result from alow count in a shallower region), white blood cell differentiation,and/or to detect more rare forms of white blood cells. Conversely, inorder to determine mean corpuscular hemoglobin (MCH), mean corpuscularvolume (MCV), red blood cell distribution width (RDW), red blood cellmorphologic features, and/or red blood cell abnormalities, opticalmeasurements (e.g., microscopic images) may be obtained from a region ofthe sample chamber having a relatively low height, since in such regionsthe cells are relatively sparsely distributed across the area of theregion, and/or form a monolayer in which the cells are relativelysparsely distributed. Similarly, in order to count platelets, classifyplatelets, and/or extract any other attributes (such as volume) ofplatelets, optical measurements (e.g., microscopic images) may beobtained from a region of the sample chamber having a relatively lowheight, since within such regions there are fewer red blood cells whichoverlap (fully or partially) with the platelets in microscopic images,and/or in a monolayer.

In accordance with the above-described examples, it is preferable to usea region of the sample carrier having a lower height for performingoptical measurements for measuring some measurands within a sample (suchas a blood sample), whereas it is preferable to use a region of thesample carrier having a greater height for performing opticalmeasurements for measuring other measurands within such a sample.Therefore, for some applications, a first measurand within a sample ismeasured, by performing a first optical measurement upon a portion ofthe sample that is disposed within a first region of the sample carrier,and a second measurand of the same sample is measured, by performing asecond optical measurement upon a portion of the sample that is disposedwithin a second region of the sample carrier. For some applications, thefirst and second measurands are normalized with respect to each other,for example, using techniques as described in a PCT application beingfiled on even date herewith, entitled “Performing optical measurementson a sample,” which is incorporated herein by reference.

For some applications, a sample carrier as described herein is used todetermine hemoglobin concentration within an undiluted blood sampleusing green light (500 nm-600 nm). For some such applications, thenominal height of the lowest region of the sample carrier is betweengreater than 1 micrometer, and/or less than 300 micrometers (e.g., 1-300micrometers). Typically, the predefined height differences betweenregions of the sample carrier are greater than 5 micrometers and/or lessthan 500 micrometers (e.g., 5-500 micrometers). For some applications,the area of each of the regions is less than 100 square millimeters,e.g., less than 25 square millimeters, although the exact dimensionstypically depend on the substrate that is used and the fabricationmethod.

For some applications, a sample carrier as described herein isconfigured such that first and second regions of the sample chambers(which are as described hereinabove) are imaged using a microscope(e.g., by providing optical windows, as described hereinabove). For somesuch applications, the nominal height of the lowest region of the samplecarrier is between greater than 40 micrometers, and/or less than 450micrometers (e.g., 4-450 micrometers). For some applications, the areaof each of the regions that is configured to be imaged by the microscopeis less than 400 square millimeters.

Reference is now made to FIG. 6, which is a flowchart showing steps ofan algorithm that is performed, in accordance with some applications ofthe present invention. The flowchart is described with reference to thesample carrier shown in FIG. 2, but the algorithm could be applied toother embodiments of the sample carrier as described herein, mutatismutandis. For some applications, the algorithm shown in FIG. 6 is usedto determine the actual height h of region 42 of sample carrier 22. In afirst step 90, an optical measurement is received from the first,shallower region 42, and a property of the sample within the region isdetermined, the property corresponding to height h. For example, whiteblood cell count within region 42 may be measured. In a second step 92,an optical measurement is obtained from the second, deeper region 44,and a property of the sample within region 44 is determined, theproperty corresponding to height h+Δh. For example, white blood cellcount within region 44 may be measured. In a third step 94, the propertywithin the height difference Δh is determined. For example, the whiteblood cell count within the height difference may be determined bysubtracting the white blood cell count from shallower region 42 from thewhite blood cell count from deeper region 44 (assuming that the areasmeasured in both of the regions were equal). In a fourth step 96, aproperty of the sample is determined based upon the known heightdifference and the property within the height difference. For example,the white blood cell count per unit volume may be determined based uponthe white blood cell count within the height difference and the knownheight difference. In a fifth step 98, height h is calculated based uponthe determined property of the sample and the property that was obtainedin step 90 within region 42. For example, based on the white blood cellcount within region 42, and the determined white blood cell count perunit volume within the sample, the volume of region 42 is derived, basedupon which height h is derived.

For some applications, the sample as described herein is a sample thatincludes blood or components thereof (e.g., a diluted or non-dilutedwhole blood sample, a sample including predominantly red blood cells, ora diluted sample including predominantly red blood cells), andparameters are determined relating to components in the blood such asplatelets, white blood cells, anomalous white blood cells, circulatingtumor cells, red blood cells, reticulocytes, Howell-Jolly bodies, etc.

In general, it is noted that although some applications of the presentinvention have been described with respect to a blood sample, the scopeof the present invention includes applying the apparatus and methodsdescribed herein to a variety of samples. For some applications, thesample is a biological sample, such as, blood, saliva, semen, sweat,sputum, vaginal fluid, stool, breast milk, bronchioalveolar lavage,gastric lavage, tears and/or nasal discharge. The biological sample maybe from any living creature, and is typically from warm blooded animals.For some applications, the biological sample is a sample from a mammal,e.g., from a human body. For some applications, the sample is taken fromany domestic animal, zoo animals and farm animals, including but notlimited to dogs, cats, horses, cows and sheep. Alternatively oradditionally, the biological sample is taken from animals that act asdisease vectors including deer or rats.

For some applications, similar techniques to those described hereinaboveare applied to a non-bodily sample. For some applications, the sample isan environmental sample, such as, a water (e.g. groundwater) sample,surface swab, soil sample, air sample, or any combination thereof. Insome embodiments, the sample is a food sample, such as, a meat sample,dairy sample, water sample, wash-liquid sample, beverage sample, and anycombination thereof.

Applications of the invention described herein can take the form of acomputer program product accessible from a computer-usable orcomputer-readable medium (e.g., a non-transitory computer-readablemedium) providing program code for use by or in connection with acomputer or any instruction execution system, such as computer processor28. For the purposes of this description, a computer-usable or computerreadable medium can be any apparatus that can comprise, store,communicate, propagate, or transport the program for use by or inconnection with the instruction execution system, apparatus, or device.The medium can be an electronic, magnetic, optical, electromagnetic,infrared, or semiconductor system (or apparatus or device) or apropagation medium. Typically, the computer-usable or computer readablemedium is a non-transitory computer-usable or computer readable medium.

Examples of a computer-readable medium include a semiconductor or solidstate memory, magnetic tape, a removable computer diskette, arandom-access memory (RAM), a read-only memory (ROM), a rigid magneticdisk and an optical disk. Current examples of optical disks includecompact disk-read only memory (CD-ROM), compact disk-read/write (CD-R/W)and DVD.

A data processing system suitable for storing and/or executing programcode will include at least one processor (e.g., computer processor 28)coupled directly or indirectly to memory elements (e.g., memory 30)through a system bus. The memory elements can include local memoryemployed during actual execution of the program code, bulk storage, andcache memories which provide temporary storage of at least some programcode in order to reduce the number of times code must be retrieved frombulk storage during execution. The system can read the inventiveinstructions on the program storage devices and follow theseinstructions to execute the methodology of the embodiments of theinvention.

Network adapters may be coupled to the processor to enable the processorto become coupled to other processors or remote printers or storagedevices through intervening private or public networks. Modems, cablemodem and Ethernet cards are just a few of the currently available typesof network adapters.

Computer program code for carrying out operations of the presentinvention may be written in any combination of one or more programminglanguages, including an object-oriented programming language such asJava, Smalltalk, C++ or the like and conventional procedural programminglanguages, such as the C programming language or similar programminglanguages.

It will be understood that blocks of the flowcharts shown in FIGS. 5 and6 and combinations of blocks in the flowcharts, can be implemented bycomputer program instructions. These computer program instructions maybe provided to a processor of a general-purpose computer, specialpurpose computer, or other programmable data processing apparatus toproduce a machine, such that the instructions, which execute via theprocessor of the computer (e.g., computer processor 28) or otherprogrammable data processing apparatus, create means for implementingthe functions/acts specified in the flowcharts and/or algorithmsdescribed in the present application. These computer programinstructions may also be stored in a computer-readable medium (e.g., anon-transitory computer-readable medium) that can direct a computer orother programmable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablemedium produce an article of manufacture including instruction meanswhich implement the function/act specified in the flowchart blocks andalgorithms. The computer program instructions may also be loaded onto acomputer or other programmable data processing apparatus to cause aseries of operational steps to be performed on the computer or otherprogrammable apparatus to produce a computer implemented process suchthat the instructions which execute on the computer or otherprogrammable apparatus provide processes for implementing thefunctions/acts specified in the flowcharts and/or algorithms describedin the present application.

Computer processor 28 is typically a hardware device programmed withcomputer program instructions to produce a special purpose computer. Forexample, when programmed to perform the algorithms described withreference to FIGS. 5 and 6, computer processor 28 typically acts as aspecial purpose sample-analysis computer processor. Typically, theoperations described herein that are performed by computer processor 28transform the physical state of memory 30, which is a real physicalarticle, to have a different magnetic polarity, electrical charge, orthe like depending on the technology of the memory that is used.

The apparatus and methods described herein may be used in conjunctionwith apparatus and methods described in any one of the following patentapplications, all of which are incorporated herein by reference:

-   US 2012/0169863 to Bachelet;-   US 2014/0347459 to Greenfield;-   US 2015/0037806 to Pollak;-   US 20150316477 to Pollak;-   US 20160208306 to Pollak;-   US 20160246046 to Yorav Raphael;-   US 20160279633 to Bachelet;-   WO 16/030897 to Yorav Raphael;-   WO 17/046799 to Eshel;-   WO 17/168411 to Eshel.

There is provided, in accordance with some applications of the presentinvention, the following inventive concepts:

Inventive concept 1. A method for performing optical measurements on abiological sample, the method comprising:

providing a sample carrier that comprises one or more sample chambersconfigured to house the sample, the one or more sample chambers definingat least first and second regions thereof, a height of the one or moresample chambers varying between the first and second regions;

categorizing the biological sample;

placing the sample into the one or more sample chambers; and

based upon the categorization of the biological sample, selecting one ofthe first and second regions upon which to perform optical measurementsfor measuring a given measurand.

Inventive concept 2. The method according to inventive concept 1,wherein categorizing the sample comprises receiving an indication of thecategorization of the sample.Inventive concept 3. The method according to inventive concept 1,wherein categorizing the sample comprises categorizing the sample basedupon a density of one or more components within the sample.Inventive concept 4. The method according to inventive concept 1,wherein categorizing the sample comprises categorizing the sample basedupon a surface density of one or more components within a monolayerformed by the sample.Inventive concept 5. The method according to inventive concept 1,wherein categorizing the sample comprises categorizing the sample basedupon a concentration of one or more components within the sample.Inventive concept 6. The method according to inventive concept 1,wherein categorizing the sample comprises categorizing the sample basedupon a count of one or more components within the sample.Inventive concept 7. The method according to inventive concept 1,wherein categorizing the sample comprises measuring a parameter of thesample selected from the group consisting of: optical absorption,transmittance, fluorescence, and luminescence, by performing apreliminary optical measurement upon the sample.Inventive concept 8. The method according to inventive concept 1,wherein categorizing the sample comprises performing microscopic imagingupon the sample.Inventive concept 9. The method according to inventive concept 1,wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the given measurand comprisesselecting one of the first and second regions upon which to performcounting of a given component within the sample, by performingmicroscopic imaging upon the region.Inventive concept 10. The method according to inventive concept 1,wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the given measurand comprisesselecting one of the first and second regions upon which to measure aconcentration of a given component within the sample, by measuring aparameter selected from the group consisting of: optical absorption,transmittance, fluorescence, and luminescence.Inventive concept 11. The method according to any one of inventiveconcepts 1-10, wherein:

the one or more sample chambers define at least first, second, and thirdregions thereof, a height of the one or more sample chambers varyingbetween each of the first, second, and third regions in a predefinedmanner; and

based upon the identified property of the biological sample, selectingtwo out of the first, second, and third regions upon which to perform,respective, first and second optical measurements for measuring thegiven measurand.

Inventive concept 12. The method according to inventive concept 11,further comprising:

performing the, respective, first and second optical measurements uponthe selected two regions; and

measuring the given measurand by using a relationship between the firstoptical measurement, the second optical measurement, and the predefinedvariation in height between the selected two regions.

Inventive concept 13. The method according to any one of inventiveconcepts 1-10, wherein the biological sample includes a blood sample,and wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the given measurand comprisesselecting one of the first and second regions upon which to performoptical measurements for measuring a given measurand of the bloodsample.Inventive concept 14. The method according to inventive concept 13,wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the given measurand comprisesselecting one of the first and second regions upon which to measure aconcentration of a given component within the blood sample, by measuringa parameter selected from the group consisting of: optical absorption,optical transmittance, fluorescence, and luminescence.Inventive concept 15. The method according to inventive concept 13,wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the given measurand comprisesselecting one of the first and second regions upon which to performcounting of a given component within the blood sample, by performingmicroscopic imaging upon the region.Inventive concept 16. Apparatus for determining a property of abiological sample, the apparatus comprising:

a sample carrier that comprises one or more sample chambers configuredto house the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions; and

a computer processor configured to:

-   -   categorize the biological sample, and    -   based upon the categorization of the biological sample, select        one of the first and second regions upon which to perform        optical measurements for measuring a given measurand of the        biological sample.        Inventive concept 17. A computer software product, for use with        a biological sample that is placed within a sample carrier that        comprises one or more sample chambers configured to house the        sample, the one or more sample chambers defining at least first        and second regions thereof, a height of the one or more sample        chambers varying between the first and second regions, the        computer software product comprising a non-transitory        computer-readable medium in which program instructions are        stored, which instructions, when read by a computer cause the        computer to perform the steps of:

categorizing the biological sample; and

based upon the categorization of the biological sample, selecting one ofthe first and second regions upon which to perform optical measurementsfor measuring a given measurand of the biological sample.

Inventive concept 18. Apparatus for performing optical measurements on abiological sample, the apparatus comprising:

a sample carrier that comprises one or more sample chambers configuredto house the sample,

the one or more sample chambers defining at least first, second, andthird regions thereof, a height of the one or more sample chambersvarying between each of the first, second, and third regions in apredefined manner.

Inventive concept 19. A method for performing optical measurements on abiological sample, the method comprising:

providing a sample carrier, the sample carrier including one or moresample chambers configured to house the sample, the one or more samplechambers defining at least first and second regions thereof, a height ofthe one or more sample chambers varying between the first and secondregions;

placing the sample into the one or more sample chambers;

measuring a first measurand, by performing a first optical measurementupon a portion of the sample that is disposed within the first region;and

measuring a second measurand, by performing a second optical measurementupon a portion of the sample that is disposed within the second region.

Inventive concept 20. The method according to inventive concept 19,wherein the biological sample includes a blood sample, wherein measuringthe first measurand comprises measuring a first measurand of the bloodsample performing the first optical measurement upon a portion of theblood sample that is disposed within the first region, and whereinmeasuring the second measurand comprises measuring a second measurand ofthe blood sample by performing a second optical measurement upon aportion of the sample that is disposed within the second region.Inventive concept 21. The method according to inventive concept 20,wherein measuring the first measurand of the blood sample comprisesdetermining a count of a first component within the blood sample byperforming microscopic imaging upon the portion of the sample that isdisposed within the first region, and wherein measuring the secondmeasurand of the blood sample comprises determining a count of a secondcomponent within the blood sample by performing microscopic imaging uponthe portion of the sample that is disposed within the second region.Inventive concept 22. The method according to inventive concept 20,wherein measuring the first measurand of the blood sample comprisesmeasuring a concentration of a first component within the blood sample,by performing, upon the portion of the sample that is disposed withinthe first region, an optical measurement of a parameter selected fromthe group consisting of: optical absorption, transmittance,fluorescence, and luminescence.Inventive concept 23. The method according to inventive concept 22,wherein measuring the second measurand of the blood sample comprisesmeasuring a concentration of a second component within the blood sample,by performing, upon the portion of the sample that is disposed withinthe second region, an optical measurement of a parameter selected fromthe group consisting of: optical absorption, transmittance,fluorescence, and luminescence.Inventive concept 24. The method according to inventive concept 22,wherein measuring the second measurand of the blood sample comprisesdetermining a count of a second component within the blood sample byperforming microscopic imaging upon the portion of the sample that isdisposed within the second region.Inventive concept 25. Apparatus for determining a property of abiological sample, the apparatus comprising:

a sample carrier that comprises one or more sample chambers configuredto house the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions; and

a computer processor configured to:

-   -   measure a first measurand, by receiving a first optical        measurement performed upon a portion of the sample that is        disposed within the first region; and    -   measure a second measurand, by receiving a second optical        measurement performed upon a portion of the sample that is        disposed within the second region.        Inventive concept 26. A computer software product, for use with        a biological sample that is placed within a sample carrier that        comprises one or more sample chambers configured to house the        sample, the one or more sample chambers defining at least first        and second regions thereof, a height of the one or more sample        chambers varying between the first and second regions, the        computer software product comprising a non-transitory        computer-readable medium in which program instructions are        stored, which instructions, when read by a computer cause the        computer to perform the steps of:

measuring a first measurand, by receiving a first optical measurementperformed upon a portion of the sample that is disposed within the firstregion; and

measuring a second measurand, by receiving a second optical measurementperformed upon a portion of the sample that is disposed within thesecond region.

Inventive concept 27. A method for performing optical measurements on abiological sample, the method comprising:

providing a sample carrier that comprises one or more sample chambersconfigured to house the sample, the one or more sample chambers definingat least first and second regions thereof, a height of the one or moresample chambers varying between the first and second regions;

categorizing a measurand of the biological sample that is to bemeasured;

placing the sample into the one or more sample chambers; and

based upon the categorization of the measurand, selecting one of thefirst and second regions upon which to perform optical measurements formeasuring the identified measurand.

Inventive concept 28. The method according to inventive concept 27,wherein the biological sample includes a blood sample, whereincategorizing the measurand of the biological sample that is to bemeasured comprises categorizing a measurand of the blood sample that isto be measured.Inventive concept 29. The method according to inventive concept 28,wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the identified measurandcomprises selecting one of the first and second regions upon which tomeasure a concentration of a given component within the blood sample, bymeasuring a parameter selected from the group consisting of: opticalabsorption, transmittance, fluorescence, and luminescence.Inventive concept 30. The method according to inventive concept 28,wherein selecting one of the first and second regions upon which toperform optical measurements for measuring the identified measurandcomprises selecting one of the first and second regions upon which toperform microscopic imaging.Inventive concept 31. Apparatus for determining a property of abiological sample, the apparatus comprising:

a sample carrier that comprises one or more sample chambers configuredto house the sample, the one or more sample chambers defining at leastfirst and second regions thereof, a height of the one or more samplechambers varying between the first and second regions; and

a computer processor configured to:

-   -   categorize a measurand of the biological sample that is to be        measured, and    -   based upon the categorization of the measurand, select one of        the first and second regions upon which to perform optical        measurements for measuring the identified measurand.        Inventive concept 32. A computer software product, for use with        a biological sample that is placed within a sample carrier that        comprises one or more sample chambers configured to house the        sample, the one or more sample chambers defining at least first        and second regions thereof, a height of the one or more sample        chambers varying between the first and second regions, the        computer software product comprising a non-transitory        computer-readable medium in which program instructions are        stored, which instructions, when read by a computer cause the        computer to perform the steps of:

categorizing a measurand of the biological sample that is to bemeasured; and

based upon the categorization of the measurand, selecting one of thefirst and second regions upon which to perform optical measurements formeasuring the identified measurand.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed hereinabove. Rather, the scope of the present inventionincludes both combinations and subcombinations of the various featuresdescribed hereinabove, as well as variations and modifications thereofthat are not in the prior art, which would occur to persons skilled inthe art upon reading the foregoing description.

1. Apparatus for determining a property of a biological sample, theapparatus comprising: a sample carrier that comprises: a glass substrateconfigured to define a first surface of a sample chamber that isconfigured to receive the sample; a plastic substrate configured todefine a second surface of the sample chamber, a height of the samplechamber at each location within the sample chamber being defined by agap between the first surface and the second surface; and an adhesivethat adheres the glass substrate to the plastic substrate, the plasticsubstrate being shaped such that the sample chamber defines a firstregion and a second region, with the sample chamber defining apredefined variation in height between the first region and the secondregion.
 2. The apparatus according to claim 1, wherein the plasticsubstrate is shaped such that the height of the chamber varies betweenthe first and second regions in a predefined stepped manner.
 3. Theapparatus according to claim 1, wherein the plastic substrate is shapedsuch that the height of the chamber varies between the first and secondregions in a predefined gradual manner.
 4. The apparatus according toclaim 1, wherein the adhesive comprises a pressure-sensitive adhesive.5. The apparatus according to claim 1, wherein the plastic substrate isshaped such that the sample chamber defines at least first, second, andthird regions, the height of the sample chamber varying between each ofthe first, second, and third regions in a predefined manner.
 6. Theapparatus according to claim 1, further comprising a computer processorconfigured to: receive data relating to a first optical measurement thatis performed upon a portion of the sample that is disposed within thefirst region, receive data relating to a second optical measurement thatis performed upon a portion of the sample that is disposed within thesecond region, and determine the property of the sample by using arelationship between the first optical measurement, the second opticalmeasurement, and the predefined variation in height between the firstregion and the second region.
 7. The apparatus according to claim 6,further comprising a microscope, wherein the computer processor isconfigured to receive the data relating to at least one of the first andsecond optical measurements by receiving imaging data from themicroscope.
 8. The apparatus according to claim 6, wherein the computerprocessor is configured to receive the data relating to at least one ofthe first and second optical measurements by receiving data relating toa parameter selected from the group consisting of: optical absorption,transmittance, fluorescence, and luminescence.
 9. The apparatusaccording to claim 6, wherein the computer processor is configured todetermine the property of the sample by determining a property selectedfrom the group consisting of: a density of a component of the sample, aconcentration of a component of the sample, and a count of a componentof the sample.
 10. The apparatus according to claim 6, wherein thecomputer processor is configured to determine an absolute height of thesample chamber within at least one of the first and second regions,using the relationship between the first optical measurement, the secondoptical measurement, and the predefined variation in height between thefirst region and the second region.
 11. The apparatus according to claim6, wherein the computer processor is configured to determine theproperty of the sample, by: subtracting a parameter derived from thefirst optical measurement from a parameter derived from the secondoptical measurement; and determining the property of the sample, basedupon a relationship between a result of the subtracting and thepredefined variation in height between the first region and the secondregion.
 12. The apparatus according to claim 6, wherein the computerprocessor is configured to determine the property of the sample, by:dividing a parameter derived from the second optical measurement by aparameter derived from the first optical measurement; and determiningthe property of the sample, based upon a relationship between a resultof the dividing and the predefined variation in height between the firstregion and the second region
 13. The apparatus according to claim 6,wherein the biological sample includes a blood sample, and wherein thecomputer processor is configured to determine the property of thebiological sample by determining a property of the blood sample.
 14. Theapparatus according to claim 13, wherein the computer processor isconfigured to determine the property of the sample by determining aproperty selected from the group consisting of: a concentration of agiven component within the blood sample, a count of a given componentwithin the blood sample, and a density of a given component within theblood sample.
 15. The apparatus according to claim 6, wherein theplastic substrate is shaped such that the sample chamber defines atleast first, second, and third regions, the height of the sample chambervarying between each of the first, second, and third regions in apredefined manner.
 16. The apparatus according to claim 15, wherein thecomputer processor is configured to: receive data relating to a thirdoptical measurement that is performed upon a portion of the sample thatis disposed within the third region; and to determine the property ofthe sample, by performing statistical analysis with respect to thefirst, second, and third optical measurements, and the predefinedvariation in height between the first, second, and third regions. 17.The apparatus according to claim 15, wherein the computer processor isconfigured to: determine a signal level of the biological sample, andbased upon the determined signal level, select two out of the first,second, and third regions upon which to perform, respectively, the firstand second optical measurements.
 18. A method for determining a propertyof a biological sample, the method comprising: placing the sample intothe a sample chamber of a sample carrier, that includes: a glasssubstrate configured to define a first surface of a sample chamber thatis configured to receive the sample, a plastic substrate configured todefine a second surface of the sample chamber, a height of the samplechamber at each location within the sample chamber being defined by agap between the first surface and a second surface, and an adhesive thatadheres the glass substrate to the plastic substrate, the plasticsubstrate being shaped such that the sample chamber defines a firstregion and a second region, with the sample chamber defining apredefined variation in height between the first region and the secondregion; performing a first optical measurement upon a portion of thesample that is disposed within the first region; performing a secondoptical measurement upon a portion of the sample that is disposed withinthe second region; and determining the property of the sample by using arelationship between the first optical measurement, the second opticalmeasurement, and the predefined variation in height between the firstregion and the second region.
 19. A method comprising: manufacturing asample carrier that defines a sample chamber for housing a biologicalsample, by placing an adhesive between a glass substrate and a plasticsubstrate; and coupling the glass substrate to the plastic substrate byapplying pressure to the adhesive, such that the glass substrate definesa first surface of the sample chamber and the plastic substrate definesa second surface of the sample chamber, a height of the sample chamberat each location within the sample chamber being defined by a gapbetween the first surface and a second surface, the plastic substratebeing shaped such that the sample chamber defines a first region and asecond region, with the sample chamber defining a predefined variationin height between the first region and the second region.
 20. The methodaccording to claim 19, wherein the adhesive includes apressure-sensitive adhesive and, wherein coupling the glass substrate tothe plastic substrate by applying pressure to the adhesive comprisesapplying pressure to the pressure-sensitive adhesive.